(i) Field of the Invention
The present invention pertains to immunogenic polypeptides, which comprise at least an epitope recognized by a protective monoclonal antibody having a high affinity and a high specificity for a surface polysaccharide of a pathogenic microorganism. The polypeptides induce an immune response in vivo against the pathogenic microorganism. The invention also relates to methods for selecting such immunogenic polypeptides, and also immunogenic or vaccinal compositions containing the polypeptides.
(ii) Description of the Related Art
Throughout this application various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Polysaccharide molecules have been shown to be present at the surface of numerous pathogenic microorganisms. Some of these polysaccharide molecules have been depicted to protect the infecting pathogenic organism from the immune system of the infected mammalian host.
The initial immunologic response to administration of a capsular polysaccharide is the production of antibodies of the IgM class, which persist for relatively short periods (Beuvery et al., 1982; Beuvery et al., 1983). A similar response is manifested after the same capsular antigen is injected a second time (Kxc3xa4yhty et al., 1984). Absence of a booster response indicates the lack of xe2x80x9cimmunologic memoryxe2x80x9d, attributes of a thymus-independent antigen.
The production of polysaccharides by bacteria has been recognized for a long time and a number of bacteria, including pneumococci, streptococci, staphylococci, menigococci, ASalmonella, Shigella, Haemophilus influenza, Escherichia coli, Kilebsiella pneumoniae and Bacteroides fragilis, are frequent causes of illness in man.
The bacterial cell wall is not the sole pathogenic organism component that contains polysaccharide antigens that are considered important determinants for inducing an immune response. A lot of viruses, such as rotaviruses (Hoshino et al., 1994), parainfluenza viruses (Ray et al., 1986; Tsurudome et al., 1989, Henrickson, 1991; Kasel et al., 1984), influenza viruses (Murphy et al., 1990; Tamura et al., 1996; Ada et al., 1986; Tamura et al., 1990; Tamura et al., 1991) or immunodeficiency viruses (FIV, HIV etc.) and fungi also express polysaccharide antigens at their surface, notably under the form of highly glycosylated proteins.
Immunodeficiency viruses, like FIV or HIV, all express envelope glycoproteins (gp 120 for HIV-1, gp 125 for HIV-2) at their surfaces. These envelope glycoproteins have been shown to be deeply involved in virus entry into target cells of the host, specifically the V3 loop domain of these external glycoproteins.
Pathogenic fungi, like some strains of Candida albicans or Neurospora crassa, also express polysaccharide antigenic determinants involved in the immune response of the host (Reiss, 1986).
The main targets of the protective immune response against bacterial infection are the capsular polysaccharide as well as the Oxe2x80x94Ag carbohydrate moiety of the LPS (for a review, see Austrian, 1985). Carbohydrate antigens are T-cell independent, inducing weak antibody responses associated with the lack of a strong B cell memory response (Bondada et al., 1994). Vaccine strategies have thus been mainly focused on the development of either polysaccharide-protein conjugates or anti-idiotype vaccines based on mimicking the carbohydrate structure (Lucas, 1994). The difficult steps of the former approach are the purification of the polysaccharide (especially-when starting from LPS, which must be devoid of any residual lipid A-related endotoxic activity), and the loss of immunogenicity of the carbohydrate moiety during coupling to the protein carrier. Carbohydrate synthesis may diminish the problems associated with antigen purification, but nonetheless remains a limited solution due to the overall difficulties of carbohydrate chemistry.
The fact that the surface polysaccharide antigens of pathogenic microorganisms, and in particular the antigenic capsular polysaccharide of bacteria, seem to induce predominantly a T cell independent immune response renders these isolated or chemically synthesized antigens less valuable to use for inducing a protective immune response in the infected host.
Moreover, the synthesis of such polysaccharide antigen molecules at an industrial and commercial scale is difficult and very costly as compared with the synthesis of protein and peptide antigen compounds that are the active principals of the conventional vaccine compositions.
Thus, there is a need in the art to design protein or peptide molecules that are able to immunologically mimic the antigenic polysaccharide, specifically that are able to induce a strong and protective immune response to the corresponding pathogenic organism.
One strategy, based on the mimicry of carbohydrate antigens by anti-idiotype antibodies, is not a simple alternative to the use of the polysaccharide antigen itself, since obtaining these antibodies is relatively time-consuming, and their use in humans is still a matter of debate. Therefore, polysaccharide-protein conjugates remain, despite difficulties, the only viable strategy for human vaccination against bacterial polysaccharidic antigens investigated until now.
As the anti-idiotype antibody molecule in its entirety is unsuitable for repeated immunization, the characterization and use of its CDRs as immunogenic peptides to elicit anti-carbohydrate antibodies has recently been reported (Weternick et al., 1995), representing an additional complication. In comparison, obtaining peptide mimics using phage display technology is quite straightforward.
Over the last few years phage-displayed peptide libraries have been widely screened with antibodies as well as non-antibody molecules leading to the identification of new ligands that do not necessarily resemble the natural ones, but display similar binding capacity (for reviews see Scott et al., 1994; Cortese et al., 1995; Felici et al., 1995; Daniels et al., 1996).
The identification of peptides that mimic carbohydrate structures has also been reported (Oldenburg et al., 1992; Scott et al., 1992, Hoess et al., 1993, Bianchi et al., 1995; Bonnycastle et al., 1996; Valadon et al., 1996). This approach might be an alternative to the use of anti-idiotypic antibodies as mimics (Westerinck et al., 1995).
In particular, Valadon et al (1996) have used phage-displayed hexa- or deca-peptide libraries in order to select peptides binding to a monoclonal antibody, Mab 2H1, directed against the glucuronoxylomannan (GXM) capsular polysaccharide from Cryptococcus neoformans. These authors have selected about 35 different peptides that bind to the 2H1 anti-GXM monoclonal antibody. These peptides gathered in four different motifs, the peptides belonging to one specific motif exhibiting a significant homology (Tables 1 and 3). Further, these authors have immunized mice with some of the selected peptides (namely PA1, P601E, and P514), but have elicited only a small anti-GXM response, although they have stimulated the production of antibodies that have the 2H1 idiotype (unpublished results of the authors). There is no need to say that Valadon et al., in failing to obtain antibodies to the initial polysaccharide antigen with the selected hexa- or deca-peptides, have also failed to obtain any protective antibody against glucuronoxylomannan of Cryptococcus neoformans. 
One explanation for the failure of Valadon et al. to select random peptides inducing a significant immune response against glucuronoxylomannan of C. neoformans lies-probably in the weak specificity of the initial anti-GXM monoclonal antibody (2H1) used by these authors, which did not confer good selectivity properties in the screening steps of the candidate peptides expressed by the phage clones of the hexa- or decapeptide libraries, although this particular point is not discussed in Valadon et al.""s article. The weak specificity of the 2H1 monoclonal antibody used by Valadon et al. may be deduced from the fact that three to four rounds of selection screening has been necessary in order to select clones expressing candidate peptide mimics.
Thus, the immunogenicity of phage-displayed peptides that mimic the carbohydrate structures involved in the protective immune response against pathogens has not been reported so far. Consequently, the availability of carbohydrate peptide mimics that are able to induce a protective immune response against a pathogenic organism is a goal that had, to date, never been reached.
Consequently, the present inventors have investigated whether random peptides selected through such a strategy could act as immunogenic mimics able to induce anti-carbohydrate antibodies. The pathogen S. flexneri has been selected as a particular embodiment of the present invention, although it will be understood that the invention is not limited to this embodiment.
The inventors have recently reported that a monoclonal antibody of the IgA type directed against a serotype-specific epitope of the Oxe2x80x94Ag, mIgA C5, and present in local secretions before infection, confers protection, thus showing the fundamental role played by both the carbohydrate Oxe2x80x94Ag (especially the serotype-specific determinants) and the local humoral response against this pathogen (Phalipon et al., 1995).
More particularly, the illustrative embodiment of this invention is based on the repeated saccharidic unit of the Oxe2x80x94Ag of S. flexneri. The structure of this saccharidic unit is shown in FIG. 1.
With reference to FIG. 1, the repeated saccharidic unit of serotype 5a is shown in FIG. 1(a) and the repeated saccharidic unit of serotype 2A is shown in FIG. 1(b). The saccharidic unit is surrounded, and xe2x80x9cnxe2x80x9d indicates that it is repeated n times to constitute the Oxe2x80x94Ag. The GlcNAc and Rha residues outside the surrounding are part of the (nxe2x88x921) and (n+1) units, respectively.
Using the mIgA C5 monoclonal antibody as well as the monoclonal antibody mIgA I3, both specific for the O-antigen (Oxe2x80x94Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and both protective against homologous infection, two phage-displayed nonapeptide libraries were screened in order to select specific random peptides that are recognized with a high specificity and a high affinity by the monoclonal antibodies. The random peptides were found to be capable of inducing a protective immune response to the pathogen in animals, specifically in mice.
These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides. Immunization of mice with one of the mimotopes can confer protection against subsequent infection. Therefore, the results indicate a new technique for the development of anti-polysaccharide vaccines.
Thus, the present invention provides an immunogenic polypeptide, which comprises an epitope recognized by a protective monoclonal antibody having a high affinity and a high specificity for a surface polysaccharide of a pathogenic microorganism. The polypeptide induces an immune response in vivo against the pathogenic organism. More particularly, the immunogenic peptide of the invention defined herein induces a protective humoral and/or cellular immune response against the pathogenic organism.
This invention also provides a purified polynucleotide coding for an immunogenic polypeptide as defined herein.
The invention is also directed to a method for selecting an immunogenic polypeptide as defined herein, comprising selecting, among a random peptide library, pertinent peptides that bind with a high affinity to a specifically chosen monoclonal antibody directed against a surface polysaccharide of a pathogenic microorganism, then characterizing the selected polypeptide(s) and ensuring that the selected polypeptide(s) induce a protective immune response in a mammal host against the pathogenic microorganism.
The invention also provides an immunogenic composition comprising an immunogenic polypeptide or a purified polynucleotide according to the invention.
This invention also provides a polyclonal or a monoclonal antibody, which is directed against an immunogenic polypeptide according to the invention.